This project focuses on the roles of human hyaluronidase-1 (hHyal-1) and hyaluronidase-2 (hHyal-2) in the mediation of the biological functions assumed by the extracellular matrix polysaccharide hyaluronan (HA). HA serves as the "glue" that binds cells together. In order for cells to move in or out of the matrix, HA must be fragmented by hHyals. Repair of injured tissue requires cell movement within the matrix, as does the escape of an abnormal cell (metastasis). HA fragments are detected by neighboring cells, and an intracellular response occurs via signal transduction pathways. The signal received is dependent on the size of the HA fragments as produced by the Hyals. Of particular medical importance, hHyal-1 and hHyal-2 play key roles in tissue inflammation and in cancer, and therefore are excellent targets for the development of novel anti-cancer and anti-inflammatory therapeutics. Until recently, structure-function studies of these enzymes have not been possible owing to the technical challenge associated with high yield production of the active N-glycosylated hHyals. A method for hHyal-1 and hHyal-2 preparation has been recently developed in the Herzberg lab and the crystal structure of hHyal-1 has been determined. The stage is now set to employ in vitro methods to accurately define hHyal-1 and hHyal-2 catalytic function and inhibition, and their interactions with protein partners, CD44 - the hyaluronan biding protein, RON receptor tyrosine kinase that is regulated by hHyal-2, and Jaagsiekte sheep retrovirus envelope protein that activates RON and uses hHyal-2 to attach to the host cell. The research plan set forth in this proposal will illuminate at the atomic level the molecular mechanisms underlying the complex cellular processes controlled by the hyaluronidases and will provide the structural foundation for the development of new therapeutics. PUBLIC HEALTH RELEVANCE: Hyaluronidase-1 and hyaluronidase-2 are crucial to the integrity and functioning of the extracellular matrix through their enzymatic activity that controls the turnover of the extracellular polysaccharide hyaluronan (HA). The HA turnover is delicately balanced and heightened hyaluronidase activity leads to inflammatory diseases and cancer progression and invasion. This project seeks to characterize the human hyaluronidases and their interactions with cellular partner proteins in vitro to shed light on their in vivo function and to lay the foundation for development of the hyaluronidases as anti cancer and anti inflammatory drug targets.